The Preliminary Study On A Functional Peptide Of FnBPA Protein Mediating Staphylococcus Aureus Invasion

Staphylococcus aureus is a common Gram positive pathogens. It can readily survive in various tissues and cause a wide spectrum of diseases. S. aureus is classically considered as an extracellular pathogen. However, it is now recognized that S.aureus can invade a variety of non-professional phagocytes, e.g. human endothelial cells, a bovine mammary epithelial cell line and murine broblasts in a fibronectin-binding proteins(FnBP)-dependent manner.The invasion process has been proposed to occur through a bridging model where fibronectin (Fn) is bound by S. aureus either of the two known forms of FnBPs, FnBPA or FnBPB as well as host cell integrins. The known fibronectin binding site of FnBPA could be mapped to the region of D domain, with 3 consecutive repeats of 37 or 38 amino acids (designated D₁, D₂ and D₃) plus one incomplete repeat (D₄). It is supposed that one complete repeat represents a binding site. If that is so, truncated D4 repeat will be functionally redundant, and the stereospecific blockade between FnBPA and Fn will lead to actually reduced binding sites. We suspect that FnBPA D repeats may contain more binding sites to get efficient binding. That is to say, not only complete repeat bind Fn, but also certain truncated repeat and combinations of partial repeats bind Fn. Our objective is to testify the above presumption and find new Fn-binding sites in D domain of FnBPA.Firstly, We reproduce a S. aureus invasion of 293 cell model according to reports, and screen a more invasive strain of 04018. Secondly, we cloned the gene fragment of interesting peptide that comprised of C-terminal D1 and N-terminal D2 (FnBPA-Dlc/D2n) from the genome of 04018. The peptide is then expressed in E. coli in GST-fusion form by use of DNA recombinant technique and genetic engineering. The peptide of the fusion protein can bind fibronectin by ELISA assay, and inhibit the internalization of S.aureus by 293 cells efficiently by invasion assay. The 50% inhibitory dose of the fusion protein is 20μg/ml, which is about 2.5μg/ml peptide on an equimolar basis.To conclude, we find a new fibronetin-binding site in FnBPA D domain. The fusion peptide of C-terminal D₁ and N-terminal D₂ can bind fibronectin as effective as a complete repeat. It is therefore presumed that either the actually binding unite in D domain is shorter than a complete rpeate, or D domain contains more Fn-binding sites than supposed. Our work also provides a useful basis on developing new therapeutic and vaccine strategies for S. aureus invasion of cells.